1. Field of the Invention
This invention relates generally to the field of diagnosing a disease by detecting gene-specific markers in a tissue sample, using RT-PCR. Specifically, the invention relates to a panel of truncated gene-specific reverse transcription primers that can enhance detection of a cancer gene in a tissue sample.
2. Background Art
Formalin-fixed, paraffin-embedded tissues (PET) are a unique source of research material with the potential of providing biological information in conjunction with known clinical outcome. Specifically, these tissues could be an ideal resource for validating newly discovered genes as diagnostic and/or prognostic molecular markers in retrospective studies. Unfortunately, RNA isolated from PET samples is considered to be a poor material for molecular analyses, since RNA is frequently degraded to 100-200 bp fragments by endogenous and exogenous ribonucleases (RNase) (1). In addition, Masuda et al have shown that RNA, and in particular the poly(A) tail of mRNA, is chemically modified, making it a poor template for cDNA synthesis (2).
Despite the technical obstacles in the analysis of PET samples, significant improvements have been made in recent years, and various studies have shown that PET samples can be used for RT-PCR analysis. The introduction of real-time RT-PCR has helped to overcome some of the difficulties of analyzing degraded RNA due to the fact that this technology has been optimized for the sensitive amplification and detection of short gene fragments (3, 1, 4). The results obtained from PET sample analysis are highly dependent on the efficiency of reverse transcription. Depending on the quality of RNA, two priming methods are commonly used in cDNA synthesis: oligo(dT) primers and random hexamers. Oligo(dT) primers anneal to the poly(A) tail of mRNA. Although this method is preferred with high quality RNA, some studies have also used it for PET analysis (5, 2). In the case of degraded mRNA, where the poly(A) tail is often fragmented and/or chemically modified (PET samples), the use of random hexamers is preferred (1, 4, 6). The major limitation of priming with random hexamers is that any RNA template, not just mRNA, can be primed.
There is a third less frequently used priming method that relies on gene-specific primers. Gene-specific reverse transcription is used to increase the specificity of the cDNA synthesis and/or enhance the detection level of low abundance RNA transcripts. Although studies have shown that gene-specific reverse transcription can increase the signal detection for a single gene (7, 8), the use of multiple gene-specific primers in a single reaction has been problematic due to the presumed formation of primer-dimers that interfere with the reverse transcription and/or subsequent PCR (9).